Conservation Genetics of Exploited Finnish Salmonid Fishes

Genetic diversity is one of the levels of biodiversity that the World Conservation Union (IUCN) has recognized as being important to preserve. This is because genetic diversity is fundamental to the future evolution and to the adaptive flexibility of a species to respond to the inherently dynamic nature of the natural world. Therefore, the key to maintaining biodiversity and healthy ecosystems is to identify, monitor and maintain locally-adapted populations, along with their unique gene pools, upon which future adaptation depends. Thus, conservation genetics deals with the genetic factors that affect extinction risk and the genetic management regimes required to minimize the risk. The conservation of exploited species, such as salmonid fishes, is particularly challenging due to the conflicts between different interest groups. In this thesis, I conduct a series of conservation genetic studies on primarily Finnish populations of two salmonid fish species (European grayling, Thymallus thymallus, and lake-run brown trout, Salmo trutta) which are popular recreational game fishes in Finland. The general aim of these studies was to apply and develop population genetic approaches to assist conservation and sustainable harvest of these populations. The approaches applied included: i) the characterization of population genetic structure at national and local scales; ii) the identification of management units and the prioritization of populations for conservation based on evolutionary forces shaping indigenous gene pools; iii) the detection of population declines and the testing of the assumptions underlying these tests; and iv) the evaluation of the contribution of natural populations to a mixed stock fishery. Based on microsatellite analyses, clear genetic structuring of exploited Finnish grayling and brown trout populations was detected at both national and local scales. Finnish grayling were clustered into three genetically distinct groups, corresponding to northern, Baltic and south-eastern geographic areas of Finland. The genetic differentiation among and within population groups of grayling ranged from moderate to high levels. Such strong genetic structuring combined with low genetic diversity strongly indicates that genetic drift plays a major role in the evolution of grayling populations. Further analyses of European grayling covering the majority of the species’ distribution range indicated a strong global footprint of population decline. Using a coalescent approach the beginning of population reduction was dated back to 1 000-10 000 years ago (ca. 200-2 000 generations). Forward simulations demonstrated that the bottleneck footprints measured using the M ratio can persist within small populations much longer than previously anticipated in the face of low levels of gene flow. In contrast to the M ratio, two alternative methods for genetic bottleneck detection identified recent bottlenecks in six grayling populations that warrant future monitoring. Consistent with the predominant role of random genetic drift, the effective population size (N_e) estimates of all grayling populations were very low with the majority of N_e estimates below 50. Taken together, highly structured local populations, limited gene flow and the small N_e of grayling populations indicates that grayling populations are vulnerable to overexploitation and, hence, monitoring and careful management using the precautionary principles is required not only in Finland but throughout Europe. Population genetic analyses of lake-run brown trout populations in the Inari basin (northernmost Finland) revealed hierarchical population structure where individual populations were clustered into three population groups largely corresponding to different geographic regions of the basin. Similar to my earlier work with European grayling, the genetic differentiation among and within population groups of lake-run brown trout was relatively high. Such strong differentiation indicated that the power to determine the relative contribution of populations in mixed fisheries should be relatively high. Consistent with these expectations, high accuracy and precision in mixed stock analysis (MSA) simulations were observed. Application of MSA to indigenous fish caught in the Inari basin identified altogether twelve populations that contributed significantly to mixed stock fisheries with the Ivalojoki river system being the major contributor (70%) to the total catch. When the contribution of wild trout populations to the fisheries was evaluated regionally, geographically nearby populations were the main contributors to the local catches. MSA also revealed a clear separation between the lower and upper reaches of Ivalojoki river system – in contrast to lower reaches of the Ivalojoki river that contributed considerably to the catch, populations from the upper reaches of the Ivalojoki river system (>140 km from the river mouth) did not contribute significantly to the fishery. This could be related to the available habitat size but also associated with a resident type life history and increased cost of migration. The studies in my thesis highlight the importance of dense sampling and wide population coverage at the scale being studied and also demonstrate the importance of critical evaluation of the underlying assumptions of the population genetic models and methods used. These results have important implications for conservation and sustainable fisheries management of Finnish populations of European grayling and brown trout in the Inari basin.

Phylogeography and population genetics of north European Atlantic salmon (Salmo salar L.)

Although abundant in the number of individuals, the Atlantic salmon may be considered as a threatened species in many areas of its native distribution range. Human activities such as building of power plant dams, offshore overfishing, pollution, clearing of riverbeds for timber floating and badly designed stocking regimes have diminished the distribution of Atlantic salmon. As a result of this, many of the historical populations both in Europe and northern America have gone extinct or are severely depressed. In fact, only 1% of Atlantic salmon existing today are of natural origin, the rest being farmed salmon. All of this has lead to a vast amount of research and many restoration programmes aiming to bring Atlantic salmon back to rivers from where it has vanished. However, many of the restoration programmes conducted thus far have been unsuccessful due to inadequate scientific research or lack of its implementation, highlighting the fact that more research is needed to fully understand the biology of this complex species. The White and Barents Seas in northwest Russia are among the last regions in Europe where Atlantic salmon populations are still stable, thus forming an important source of biodiversity for the entire European region. Salmon stocks from this area are also of immense economic and social importance for the local people in the form of fishing tourism. The main aim of this thesis was to elucidate the post-glacial history and population genetic structure of north European and particularly northwest Russian Atlantic salmon, both of which are aspects of great importance for the management and conservation of the species. Throughout the whole thesis, these populations were studied by utilizing microsatellites as the main molecular tool. One of the most important discoveries of the thesis was the division of Atlantic salmon from the White and Barents Seas into four separate clusters, which has not been observed in previous studies employing nuclear markers although is supported by mtDNA studies. Populations from the western Barents Sea clustered together with the northeast Atlantic populations into a clearly distinguishable group while populations from the White Sea and eastern Barents Sea were separated into three additional groups. This has important conservation implications as this thesis clearly indicates that conservation of populations from all of the observed clusters is warranted in order to conserve as much of the genetic diversity as possible in this area. The thesis also demonstrates how differences in population life histories within a species, migratory behaviour in this case, and in their phylogeographic origin affect the genetic characteristics of populations, namely diversity and divergence levels. The anadromous populations from the Atlantic Ocean, White Sea and Barents Sea possessed higher levels of genetic diversity than the anadromous populations form the Baltic Sea basin. Among the non-anadromous populations the result was the opposite: the Baltic freshwater populations were more variable. This emphasises the importance of taking the life history of a population into consideration when developing conservation strategies: due to the limited possibilities for new genetic diversity to be generated via gene flow, it is expected that freshwater Atlantic salmon populations would be more vulnerable to extinction following a population crash and thus deserve a high conservation status. In the last chapter of this thesis immune relevant marker loci were developed and screened for signatures of natural selection along with loci linked to genes with other functions or no function at all. Also, a novel landscape genomics method, which combines environmental information with molecular data, was employed to investigate whether immune relevant markers displayed significant correlations to various environmental variables more frequently than other loci. Indications of stronger selection pressure among immune-relevant loci compared to non-immune relevant EST-linked loci was found but further studies are needed to evaluate whether it is a common phenomenon in Atlantic salmon.

From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Tenojoen pääuoman habitaattien määrä ja merkitys lohen (Salmo salar) poikastuotannolle

Atlantin lohen (Salmo salar) kannanhoidollisia toimenpiteitä varten vaaditaan tietoa lisääntymisjoissa olevien habitaattien määrästä ja laadusta. Tässä tutkielmassa selvitettiin Tenojoen pääuoman erilaisten habitaattien määrää ja niiden merkitystä lohen poikashabitaatteina. Tenojoen pääuoma kartoitettiin lohen habitaattien kartoitukseen kehitetyllä mesohabitaatti-luokittelulla, jossa joki kartoitetaan havainnoimalla sen hydromorfologisia piirteitä, virtausnopeutta, syvyyttä ja pohjan raekokoa. Lisäksi mitattiin suojakolojen määrää ja syvyysluokkaa erilaisilla substraateilla, ja tämän perusteella arvioitiin pohjan raekoon vaikutusta suojakolojen määriin ja syvyyteen. Lohenpoikasten esiintyvyyttä erilaisilla mesohabitaateilla, syvyyksillä ja substraateilla selvitettiin koekalastamalla laajoja alueita erityisvalmisteisella sähkökoekalastusveneellä. Raekoon, syvyyden ja suojakolojen vaikutusta lohenpoikastiheyksiin testattiin lisäksi tilastollisin menetelmin käyttämällä Luonnonvarakeskuksen tekemiä sähkökoekalastuksia. Mesohabitaattikartoituksen ja veneellä tehtyjen sähkökoekalastuksien aikana virtaama Tenojoessa oli lähellä vuosittaista minimiä. Mesohabitaattikartoituksesta selvisi, että Tenojoesta valtaosa (67 %) kuului hiekkapohjaiseen ja >50 cm/s virtaavaan mesohabitaattiin. Veneellä tehdyissä sähkökoekalastuksissa näiltä hiekka-alueilta saatiin vain vähäisiä määriä lohenpoikasia, ja nekin rantavyöhykkeiltä, joilla oli kivien ja lohkareiden muodostamia suojapaikkoja. Noin 32 % Tenojoesta on pienen virtaaman aikana lohen poikashabitaattina merkittäviä, nivamaisia tai koskimaisia alueita, jotka voidaan luokitella syvyyden ja virtausnopeuden mukaan eri mesohabitaattiluokkiin. Suojakolomittauksien perusteella vallitseva raekoko vaikuttaa merkitsevästi suojakolojen määrään ja syvyyteen. Kesän vanhat (0+) ja vanhemmat (>0+) poikaset suosivat eri syvyisiä suojakoloja, ja etenkin >0+ poikasten määrä ja >10 cm syvien suojakolojen määrän välillä oli voimakas positiivinen korrelaatio (p = 0,01). Ympäristöparametrien (syvyys, virtausnopeus ja raekoko) ja poikastiheyksien välinen suhde vastasi hyvin aiempia tutkimuksia, mutta sekä kesän vanhoja (0+) ja vanhempia (>0+) poikasia havaittiin aikaisempien tutkimuksien preferenssiarvoja syvemmillä alueilla. Veneellä tehdyissä sähkökoekalastuksissa havaittiin suurempia poikastiheyksiä rannanläheisillä linjoilla verrattuna joen keskellä tehtyihin koekalastuslinjoihin, kun vertailtiin habitaattiolosuhteiltaan samankaltaisia linjoja, jotka olivat ainakin osittain joen pituussuunnassa päällekkäin.

Kylmän veden tauti kirjolohella : tapausselostus ja lääkejäämien seuranta

Eija Rimaila-Pärnänen, Varpu Hirvelä-Koski, Anneli Niemi, Jaana Seppänen ja Erkki Hernesniemi

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Annotation of Gyrodactylus salaris transcriptome

Relationship between organisms within an ecosystem is one of the main focuses in the study of ecology and evolution. For instance, host-parasite interactions have long been under close interest of ecology, evolutionary biology and conservation science, due to great variety of strategies and interaction outcomes. The monogenean ecto-parasites consist of a significant portion of flatworms. Gyrodactylus salaris is a monogenean freshwater ecto-parasite of Atlantic salmon (Salmo salar) whose damage can make fish to be prone to further bacterial and fungal infections. G. salaris is the only one parasite whose genome has been studied so far. The RNA-seq data analyzed in this thesis has already been annotated by using LAST. The RNA-seq data was obtained from Illumina sequencing i.e. yielded reads were assembled into 15777 transcripts. Last resulted in annotation of 46% transcripts and remaining were left unknown. This thesis work was started with whole data and annotation process was continued by the use of PANNZER, CDD and InterProScan. This annotation resulted in 56% successfully annotated sequences having parasite specific proteins identified. This thesis represents the first of Monogenean transcriptomic information which gives an important source for further research on this specie. Additionally, comparison of annotation methods interestingly revealed that description and domain based methods perform better than simple similarity search methods. Therefore it is more likely to suggest the use of these tools and databases for functional annotation. These results also emphasize the need for use of multiple methods and databases. It also highlights the need of more genomic information related to G. salaris.